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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2010 December 24; 285(52): 40654–40665.
Published online 2010 October 22. doi:  10.1074/jbc.M110.166041
PMCID: PMC3003364

The N-terminal Region of Twitchin Binds Thick and Thin Contractile Filaments

REDUNDANT MECHANISMS OF CATCH FORCE MAINTENANCE*

Abstract

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015–18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and Kd values of 1 and 15 μm, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the region's central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.

Keywords: Cell Motility, Contractile Protein, Molecular Motors, Protein Motifs, Protein Phosphorylation

Introduction

Catch is a mechanical state present in some invertebrate smooth muscles in which force output and high resistance to muscle lengthening are maintained for long periods of time with very low energy utilization. In the catch state, intracellular [Ca2+] is close to basal concentration (1), and redevelopment of force following unloading of the muscle is absent (2). A hypothesis to explain the long term maintenance of catch force was based on a slowing of cycling of myosin attached to actin when activation waned (see Lowy and Millman (3)). On the other hand, Ruegg proposed that catch was maintained by an independent linkage among thick filaments, possibly mediated by paramyosin (4, 5). As will be discussed below, much of the recent evidence supports the idea that catch results from an independent linkage between thick and thin filaments.

Catch force is rapidly relaxed by stimulation of serotonergic nerves (6) that results in an increase in cAMP (7) and subsequent activation of cyclic AMP-dependent protein kinase (PKA)2 (8). The protein that is phosphorylated by PKA during relaxation of catch force is twitchin, and it is the phosphorylation state of this protein that determines the presence or absence of the catch state at basal [Ca2+] (9, 10). Twitchin is so named because Caenorhabditis elegans mutants that lack the protein show nearly constant twitching of body wall muscles (11). Twitchin is a mini-titin that is composed of Ig and fibronectin domains as well as a kinase domain near the C terminus (11). The domain organization of twitchin from Mytilus (12) is shown in Fig. 1. The central portion of the molecule shares domain homology with part of the A band portion of titin, and twitchin is known to be associated with thick filaments in catch muscle (13, 14). In vitro, twitchin is phosphorylated by PKA with a stoichiometry of about 3 mol of phosphate/mol of twitchin (15). The D1 phosphorylation site is located between the 7th and 8th Ig domain in the N-terminal part of the molecule, and the D2 site is near the C terminus between the 21st and 22nd Ig domain (12, 15). Another site of phosphorylation shares a seven-amino acid sequence with the D1 site, is located in the same Ig linker region, and is referred to as DX (16). Also present in the same linker region as D1 and DX is a DFRXXL actin-binding motif (17) similar to those that mediate the binding of smooth muscle myosin light chain kinase to the thin filament (18) and myosin IIIA tail interactions with the thin filament (19).

FIGURE 1.
Twitchin molecule and portions used in experimental protocols. Green, immunoglobulin-like motif; yellow, fibronectin type III motif; blue, kinase domain. Phosphorylation sites are shown in red. The underlined regions show sequences used in experiments. ...

Using an in vitro motility assay, Yamada et al. (20) showed that mechanical aspects of the catch state can be demonstrated using thick filaments reconstituted from purified myosin, F-actin, and twitchin. They found, however, that little if any binding of twitchin to actin occurred in co-sedimentation experiments. On the other hand, Shelud'ko et al. (21) described significant twitchin-actin interactions that were greatly decreased when twitchin was phosphorylated. Their results supported our previous suggestion (22) that twitchin might provide a mechanical link between thick and thin filaments. The idea that catch force is maintained by a mechanical link not involving the high force myosin cross-bridge is also supported by studies showing that agents that block force output by myosin cross-bridges have no effect when added to permeabilized muscles in catch (23, 24) and actually increase catch force when added to activated muscles (24). Also, there is no measurable turnover of the ADP bound to myosin when catch force is relaxed by phosphorylation of twitchin (25). Insight regarding the portion of twitchin that is involved in possible tethering of thick and thin filaments was provided by the finding that a fragment of twitchin containing the D2 phosphorylation site and flanking Ig domains bound to both actin and myosin when D2 was unphosphorylated but not when phosphorylated (26). This and further studies (27) showed that this fragment of twitchin forms a phosphorylation-sensitive heterotrimeric complex with a portion of the myosin loop 2 region and actin subdomain 1. The implications of such a complex in mediating a connection of thick and thin filaments in catch are obvious.

The purpose of this study was to determine whether there are thick and thin filament binding regions in the twitchin molecule in addition to those identified in the C-terminal D2 region. The focus was on the N-terminal region containing the D1 and DX regulatory phosphorylation sites that also includes the DFRXXL actin binding motif. The specific portions of the twitchin molecule from Mytilus anterior byssus retractor muscle (ABRM) that were studied are shown in Fig. 1. Peptides ranging from 47 to 52 amino acids included the following: PEVK1, DX, D1, and D2. PEVK1 is from a PEVK-rich region of twitchin that is shorter and has a lower frequency of PEVK residues than titin from vertebrate muscle (12). This peptide was chosen because the PEVK domain from titin has been shown to interact with actin (28,30). In addition to the DX phosphorylation site, the DX peptide contains the DFRXXL motif. The D1 and the D2 peptides include the D1 and D2 phosphorylation sites, respectively, and surrounding sequences. Recombinant proteins that were studied included IGDXD1IG, DXD1, and IGD2IG. DXD1 includes all of the residues in the linker region between Ig domains containing the DFRXXL motif and the DX and D1 phosphorylation sites, whereas IGDXD1IG includes this region plus the adjoining Ig domains that are the 7th and 8th from the N terminus of twitchin. IGD2IG contains the D2 phosphorylation site near the C terminus of twitchin as well as the flanking Ig domains.

EXPERIMENTAL PROCEDURES

Peptide Synthesis and Recombinant Protein Expression and Purification

Peptides with an N-terminal fluorescein isothiocyanate label were synthesized, and gene synthesis, recombinant protein (with an N-terminal His6 tag) expression in Escherichia coli, and His tag protein purification using a nickel-iminodiacetic acid column were performed by GenScript Corp. (Piscataway, NJ). A peptide with the sequence Ac-AQNKEAETTGTHKKRKSSA-amide was obtained from New England Peptide, LLC (Gardner, MA). This peptide is based on the myosin loop 2 sequence and was shown by Funabara et al. (27) to inhibit the formation of a trimeric complex between actin, myosin, and the D2 portion of twitchin. All recombinant proteins were subjected to chromatography through a Sephadex G-25 column using an AKTA Prime Plus chromatography system (Amersham Biosciences) to effect an exchange into the solution of choice. Fig. 2, A–C, shows SDS-polyacrylamide gels containing the recombinant proteins.

FIGURE 2.
Coomassie Blue-stained SDS-polyacrylamide gels of recombinant proteins IGDXD1IG (lane A), DXD1 (lane B), and IGD2IG (lane 3). Isolated thin filaments (lane D) and thick filaments (lane E) are also shown. Myosin heavy chain (MHC), paramyosin (Para), and ...

Isolation of Thick and Thin Filaments

Thick and thin filaments were isolated from Mytilus edulis ABRM by differential centrifugation using a modification of the method of Yamada et al. (13). Briefly, muscles were treated with Triton X-100 (25), minced, and then homogenized with a Polytron homogenizer in a filament extraction solution containing 10 mm MgATP, 10 mm EGTA, 2.5 mm DTT, 1 mm free Mg2+, 10 mm PIPES, and 50 μm leupeptin, pH 7.0. The mixture was centrifuged at 700 × g for 5 min, and the supernatant was removed. This was repeated, and the supernatant was then spun at 5000 × g for 30 min. The thick filaments in the pellet were resuspended in the desired solution. The thin filament-containing supernatant was centrifuged at 134,000 × g for 5 min in an Airfuge (Beckman Coulter Inc., Fullerton, CA) to pellet any remaining thick filaments, and the supernatant was subsequently spun for 30 min at the same speed. The thin filament-containing pellet was subsequently resuspended. Fig. 2, D–E, shows SDS-polyacrylamide gels of the thin and thick filament preparations. The thin filaments consist predominantly of actin and a slightly lower molecular weight protein that shows about 20% of the staining of actin. Most of the tropomyosin was probably lost during purification (31), and as such, the thin filaments are similar to F-actin. The thick filaments consist mainly of myosin and paramyosin with only a very small actin component.

Binding of Recombinant Proteins to Thin Filaments

All binding experiments were performed at 20 °C. Determination of IGDXD1IG and DXD1 binding to thin filaments was done using a modification of the co-sedimentation technique described by Shaffer et al. (32) for measuring binding of various fragments of myosin-binding protein C to F-actin. The assay solution was based on a relaxing solution used for permeabilized muscles (24, 25). It contained 30 mm PIPES, 20 mm EGTA, 3 mm MgATP, 3 mm free Mg2+, 1 mm DTT, and 21 mm 1,6-diaminohexane-N,N,N′,N′-tetraacetic acid (HDTA), pH 6.8. Ionic strength was 202 mm. Imidazole (100 mm) was included to help maintain recombinant protein solubility. Separate studies showed that 100 mm imidazole had no effect on active force, catch force, or relaxation of catch force with the addition of cAMP in permeabilized ABRM (data not shown). The thin filaments and different concentrations of the recombinant proteins were combined and allowed to equilibrate for 30 min. This was followed by centrifugation in an Airfuge at 134,000 × g for 30 min. The supernatant was removed, and the pellet was carefully washed with a small volume of relaxing solution. The pellet was taken up in SDS sample buffer and subjected to SDS-PAGE. The gels also contained known standards of the recombinant proteins. The protein stain (Coomassie Brilliant Blue R-250) was quantified using a laser densitometer (Molecular Dynamics), and the molar ratio of recombinant protein to actin was determined for each sample. Kd and Bmax were estimated using the one-site saturation ligand binding regression program in the Systat software. In order to test the effect of phosphorylation of the recombinant proteins on the binding to the thin filament, proteins were phosphorylated by the addition of the catalytic subunit of cyclic AMP-dependent protein kinase (Calbiochem) (150 kilounits/ml) for 1 h at 20 °C. Phosphorylation was confirmed using Pro Q Diamond phosphoprotein stain (Invitrogen), and the D1 phosphorylation-dependent antibody (12) confirmed nearly 100% phosphorylation of the D1 site.

Co-sedimentation of Recombinant Proteins with Thick Filaments

IGDXD1IG and DXD1 were mixed with a relaxing solution containing thick filaments. After 1 h, the solution was centrifuged at 134,000 × g for 5 min. The pellets containing the thick filaments were carefully washed with relaxing solution and then subjected to SDS-PAGE and Western blotting. The amount of recombinant protein was quantified with a His tag antibody and compared with a control in which the thick filaments were removed from the solution prior to the addition of the recombinant proteins.

Solid Phase Binding Assay

Even with imidazole present, it was difficult to maintain the solubility of IGD2IG for the duration of the co-sedimentation experiments described above. In order to confirm the binding of IGD2IG to the thin filament and to compare it with IGDXD1IG, a solid phase binding assay modified from that described by Funabara et al. (26, 27) was used. In this case, wells in a plastic immunoplate were coated for 45 min with the recombinant proteins in a solution similar to that used for filament isolation except for the inclusion of 100 mm imidazole. The wells were blocked with 1% bovine serum albumin (diluent/blocking solution, KPL, Inc., Gaithersburg, MD) for 15 min and then washed in rigor solution used for permeabilized muscle experiments. It contained the following: 2 mm EGTA, 3 mm free Mg2+, 1 mm DTT, 30 mm PIPES, 43.2 mm HDTA, pH 6.8. Thin filaments were pelleted in an Airfuge and resuspended in rigor solution (~1.6 μm actin). Rhodamine phalloidin (Cytoskeleton, Inc.) was then added (0.3 μm). The rhodamine phalloidin-labeled thin filaments (50 μl) were added to the wells for 30 min. This was followed by several washes with rigor solution. Rhodamine fluorescence was measured in a Safire2 fluorometric plate reader (Tecan). Bovine serum albumin was used as a control in these experiments. In experiments that tested the effect of phosphorylation, the IGDXD1IG or IGD2IG in the wells was treated with the catalytic subunit of cyclic AMP-dependent protein kinase in a solution similar to rigor solution but containing 3 mm MgATP. This was followed by washing in rigor solution before the addition of the rhodamine phalloidin-labeled thin filaments. The phosphorylation of the proteins in the wells was confirmed by performing SDS-PAGE and Western blotting followed by detection using the D1 and D2 phosphorylation-sensitive antibodies.

Peptide Binding to Thin Filaments

The binding characteristics of the various peptides to thin filaments in relaxing solution were determined using a co-sedimentation assay similar to that described for the recombinant proteins. In this case, the peptide quantitation on the gel was determined by comparing the fluorescence of the peptide in a given sample on the gel (measured in a FluorChem imager, Alpha Innotech) with that from known amounts of the peptide run in the same gel. The gel was then stained with Coomassie Blue to determine the amount of actin in the sample, and the molar ratio of peptide to actin was calculated. The DX and D2 peptides were also synthesized with the DX site (Ser1011) and D2 site (Ser4316) phosphorylated. Synthesis of the D1 peptide with the D1 site (Ser1075) phosphorylated was not successful, so the peptide was phosphorylated by the addition (125 kilounits/ml to 0.16 μm peptide) of the catalytic subunit of cyclic AMP-dependent protein kinase (Calbiochem) for 1 h. Mass spectrometry showed that this resulted in phosphorylation of two sites (data not shown). Use of a phosphorylation-dependent antibody to the D1 site as described (12) confirmed phosphorylation of this site, and it is likely that the second phosphorylated site is another PKA consensus sequence in the peptide (Ser1067). It is not known if this second site is phosphorylated by PKA during the release of catch in the muscle.

Antibodies

Detection of proteins on Western blots was accomplished using one of the following primary antibodies: His6 mouse monoclonal antibody (Clontech) (1:600 dilution), rabbit anti-unphosphorylated D1 site antibody (12) (1:2000 or 1:2500 dilution), and rabbit anti-phosphorylated D2 site antibody (12) (1:200 dilution). Secondary antibodies included anti-mouse IgG peroxidase-linked whole antibody from sheep (GE Healthcare) (1:5000 dilution) and anti-rabbit IgG peroxidase-linked whole antibody from donkey (GE Healthcare) (1:5000 dilution). The Amersham Biosciences ECL Plus Western blotting detection system (GE Healthcare) was used along with a FluorChem imager.

Mechanical Measurements on Permeabilized ABRM

Muscles were dissected, permeabilized with Triton X-100, and subjected to mechanical measurements as described previously (24). The relaxing solution contained 3 mm MgATP, 5 mm phosphocreatine, 20 mm EGTA, 3 mm free Mg2+, 1 mm DTT, 30 mm PIPES, 15.6 mm HDTA, 1 mg/ml creatine phosphokinase, 0.5 mm leupeptin, pH 6.8. The [Ca2+] of the relaxing solution with no added calcium was considered to be pCa >8. The activating solution was similar to the relaxing solution, with the exception that pCa was adjusted to 6.3 by the addition of CaEGTA, whereas the total EGTA was maintained at 20 mm. In some cases, twitchin was thiophosphorylated as described (24). Thiophosphorylated twitchin is resistant to dephosphorylation, so no catch force resulting from endogenous unphosphorylated twitchin is present during these experiments. Peptides were dissolved in the appropriate permeabilized muscle solution, and the pH of this solution was adjusted to match that of the control solution. The proteins to be tested were subjected to chromatography through a Sephadex G-25 column to effect an exchange into the permeabilized muscle solution. Imidazole (100 mm) was included in all protein-containing solutions.

In some cases, the effects of peptides on catch force maintenance were tested. (±)-Blebbistatin (Calbiochem) treatment (25 μm) in pCa 6.3 was used to induce the catch state. We have previously shown that under these conditions, ATP usage by the muscle is very low, although much of the force developed before the addition of blebbistatin remains (24). All of this force is relaxed by phosphorylation of twitchin, and it is thus designated catch force.

Statistics

Statistical analyses were performed using the t test or analysis of variance programs in Sigmastat software. When necessary, two-way analysis of variance with pairwise comparisons were made using the Holm-Sidak method.

RESULTS

Binding of the Recombinant Proteins IGDXD1IG and IGD2IG to Thin Filaments

The ability of the recombinant proteins IGDXD1IG and IGD2IG to bind to thin filaments was tested in a solid phase binding experiment. Fig. 3A shows the fluorescence from rhodamine phalloidin-labeled thin filaments present after washing as a function of the amounts of the recombinant proteins added to the wells. There is a similar concentration-dependent binding of thin filaments to both IGDXD1IG and IGD2IG. Further experiments showed that the sum of the fluorescence associated with thin filament binding to IGDXD1IG (2.8 pmol) and IGD2IG (3.2 pmol) individually (13,059 ± 2356 and 9577 ± 1014, respectively) is similar to that obtained when both are added together in the well (22,565 ± 1446). This means that each of these thin filament binding domains of twitchin act independently.

FIGURE 3.
A, fluorescence from rhodamine-phalloidin-labeled thin filaments as a function of amount of IGDXD1IG (solid circles, solid line) or IGD2IG (open circles, dashed line) present in a solid phase binding assay. Fluorescence was normalized to the maximum in ...

The effect of phosphorylation of the proteins on thin filament binding was tested by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the proteins in the wells. Fig. 3, B and C, shows Western blots of the recombinant proteins treated with antibodies that show phosphorylation-dependent binding. The D1 antibody binds only to the unphosphorylated D1 site, whereas the D2 antibody binds only to the D2 phosphorylated site (12). The results confirm the phosphorylation of both the D1 and D2 sites under the conditions used. The effects of these phosphorylations on thin filament binding are shown for IGDXD1IG and IGD2IG in Fig. 3, B and C, respectively. In both cases, there is a decrease in binding of the thin filaments as a result of protein kinase A-mediated phosphorylation. These results strongly suggest that both ends of the twitchin molecule participate in the regulated tethering of twitchin from the thick to the thin filaments.

Funabara et al. (12) have shown that a peptide based on the myosin loop 2 region binds to IGD2IG and competitively inhibits the formation of a trimeric complex among actin, myosin, and IGD2IG. This suggested that the myosin loop 2 region is a participant in the formation of the trimeric complex. In order to test whether the myosin loop 2 region also plays a role in the functioning of the IGDXD1IG region of twitchin, the effect of the peptide on binding of thin filaments to IGDXD1IG was determined. Experiments similar to those described in the legend to Fig. 3A were performed with 19 pmol of IGDXD1IG in each well. Inclusion of 2.4 μm myosin loop 2 peptide in the solid phase binding assay decreased the relative binding of thin filaments to IGDXD1IG to 72 ± 6% (n = 9) of control without the peptide, and 24 μm peptide decreased binding of thin filaments to 21 ± 6% (n = 9) of the control. These data suggest that the myosin loop 2 region can modify the binding of the thin filaments to the IGDXD1IG portion of twitchin.

Recombinant Protein IGDXD1IG

The effect of the IGDXD1IG portion of twitchin on force output was tested in permeabilized muscles. At a calcium concentration (pCa 6.3) resulting in less than maximum force output, IGDXD1IG caused a significant increase in force (Fig. 4, A and B). The increase in force did not occur when the protein was phosphorylated by pretreatment with the catalytic subunit of protein kinase A (Fig. 4C). The effect of IGDXD1IG on force was dependent on concentration, as shown in Fig. 4D. The data are consistent with a binding curve having a Kd of about 13 μm, but the data set is limited by the lack of solubility of the protein at concentrations higher than about 25 μm. Experiments also showed that there was a significant increase in force output caused by IGDXD1IG when added to muscles in which force maintenance mediated by endogenous twitchin was prevented by its prethiophosphorylation (data not shown).

FIGURE 4.
Mechanical effects of IGDXD1IG at pCa 6.3. A, typical force trace from permeabilized ABRM showing the effect of IGDXD1IG (10 μm; solid line) compared with control (dashed line). B, force with (hatched bar) and without (open bar) the addition of ...

The binding of IGDXD1IG to Mytilus thin filaments was also measured in co-sedimentation studies. Fig. 5A shows a typical gel of pellets showing the concentration dependence of co-sedimentation of IGDXD1IG with thin filaments. Fig. 5B shows that unphosphorylated IGDXD1IG shows saturable binding to thin filaments with a Kd of 1.0 ± 0.3 μm and Bmax of 1.3 ± 0.1 mol/mol actin. Binding is reduced when IGDXD1IG is phosphorylated with an increase of Kd of about 3-fold with no apparent change in Bmax. Co-sedimentation of IGDXD1IG with thick filaments was also observed in experiments containing about 0.3 μm myosin heavy chain and 0.13 μm IGDXD1IG. In this case, the pellet from solutions containing the thick filaments and IGDXD1IG contained 38 ± 13% (p ≈ 0.025) more IGDXD1IG than those not containing thick filaments.

FIGURE 5.
Binding of IGDXD1IG to thin filaments. A, typical Coomassie Blue-stained SDS-polyacrylamide gels of the pellets following co-sedimentation of solutions containing thin filaments (actin = 0.8 μm) and the indicated concentrations of IGDXD1IG. B ...

Recombinant Protein DXD1

The DXD1 protein contains the portion of twitchin between N- and C-terminal IG domains surrounding the DX and D1 phosphorylation sites. The addition of the protein to permeabilized muscles at pCa 6.2 causes a small but significant increase in isometric force (Fig. 6A). DXD1 co-sediments to a small extent with thick filaments when unphosphorylated but shows no significant co-sedimentation with thick filaments when phosphorylated (Fig. 6, B and C). Quantification of the phosphorylation-dependent binding of DXD1 to thin filaments in co-sedimentation assays is shown in Fig. 7. Unphosphorylated DXD1 shows saturable binding to thin filaments with Kd = 15 ± 7 μm and Bmax = 1.0 ± 0.2 mol/mol actin. Phosphorylation of DXD1 causes a large decrease in binding to thin filaments (Fig. 7B). The extent of change in Kd and Bmax with phosphorylation could not be estimated well because of the low binding associated with the concentrations studied.

FIGURE 6.
Effect of DXD1 on force output and phosphorylation-dependent co-sedimentation of DXD1 with thick filaments. A, force with (hatched bar) and without (open bar) the addition of DXD1 (25 μm). Design is similar to that shown in Fig. 4A. n = 6 for ...
FIGURE 7.
Binding of DXD1 to thin filaments. A, typical Coomassie Blue-stained SDS-polyacrylamide gels of the pellets following co-sedimentation of solutions containing thin filaments (actin = 1.0 μm) and the indicated concentrations of DXD1. B, quantitative ...

These results on both IGDXD1IG and DXD1 show that the N-terminal portion of the twitchin molecule can interact with both actin and myosin in a phosphorylation-dependent manner and thus suggest that it plays a role in the tethering of thick and thin filaments that gives rise to catch force. This raises the possibility that a protein as small as DXD1 (consisting of 143 amino acids) may be sufficient to tether thick and thin filaments in a phosphorylation-dependent manner as has been shown for the IGD2IG portion of twitchin (27). Importantly, the phosphorylation-dependent increase in force with the addition of IGDXD1IG is consistent with the idea that the linkage of thick and thin filaments through this part of twitchin can result in the continued maintenance of some of the force generated by the myosin cross-bridge when it subsequently detaches from actin (see Ref. 24). In order to more finely map the components of twitchin that are functionally important in the mechanism of control of catch force maintenance, we studied smaller peptides of about 50 amino acids that included either the DX, D1, or D2 phosphorylation site.

Synthetic Peptide D1

The unphosphorylated synthetic D1 peptide shows significant co-sedimentation with the thin filament (Fig. 8A). The binding showed a Kd of 38 ± 11 μm and Bmax of 1.4 ± 0.2 mol/mol actin. Mass spectrometry showed that when the peptide is treated with the catalytic subunit of protein kinase A, there is a shift in mass of the peptide by 160 mass units (data not shown). This is consistent with the incorporation of two phosphates into the D1 peptide. Use of the phosphorylation-sensitive antibody to the D1 site confirmed that it is fully phosphorylated, and it is likely that the other site of phosphorylation is serine 1067, which is part of the A kinase consensus sequence RRSS. Such a phosphorylation of the D1 peptide reduced its binding to the thin filament (Fig. 8A). There is no effect of the D1 peptide (up to a concentration of about 700 μm) on isometric force at pCa 6.3 (Fig. 9). These data suggest that the region including and surrounding the D1 phosphorylation site in twitchin participates in the phosphorylation-dependent tethering of twitchin to the thin filaments. The lack of an effect of high concentrations of the D1 peptide on force suggests that this part of twitchin binds to the thin filament at a site that does not interfere with actin-myosin interaction.

FIGURE 8.
Quantitation of the co-sedimentation of peptides with thin filaments. Overall design was similar to that described in the legend to Fig. 5. All peptides had FITC attached, and fluorescence was detected on gels containing proteins in the pellets formed ...
FIGURE 9.
Effect of twitchin peptides on isometric force output. Permeabilized muscles were activated in pCa 6.3 for 12 min and then incubated in a similar pCa 6.3 solution either containing the peptide or not. The force output after 30 min in the peptide was normalized ...

Synthetic Peptide DX

The DX peptide contains a DFRXXL actin binding site motif and the DX phosphorylation site. Fig. 8B shows that there is co-sedimentation of the DX peptide with thin filaments and that the extent of this co-sedimentation is decreased when the DX site is phosphorylated. Muscles with thiophosphorylated twitchin were used in this experiment in order to directly determine the effect of the peptide on force output from myosin cross-bridge cycling rather than its effect on catch force output. When twitchin is thiophosphorylated, there is no catch force maintenance. The unphosphorylated DX peptide totally inhibits isometric force at high peptide concentrations (~700 μm) (Fig. 9). This suggests that DX interferes with either myosin binding to actin or with the low to high force transition in the myosin-actin complex. When the peptide was phosphorylated, the extent of inhibition was significantly lower (Fig. 9). The phosphorylation dependence of the inhibition of force output is consistent with decreased binding to the thin filament by the phosphorylated peptide.

Synthetic Peptide D2

Funabara et al. (26, 27) have shown that unphosphorylated IGD2IG co-sediments with both thick and thin filaments. Because the binding of the N-terminal segment of IGDXD1IG to actin seems to be mediated by sequences immediately surrounding the phosphorylation sites, we tested whether the same was true for IGD2IG. The unphosphorylated D2 peptide shows saturable binding to thin filaments (Fig. 8C) with a Kd of 31 ± 13 μm and a Bmax of 2.1 ± 0.3. It thus appears that there are two sites of D2 peptide binding for every actin monomer in the thin filament. Phosphorylation of the peptide decreases binding to a large extent (Fig. 8C), with the Bmax decreasing to 1.1 ± 0.2 without a significant change in Kd (32 ± 17 μm). The data suggest that one of the two binding sites loses its ability to bind D2 when the peptide is phosphorylated. There is a small increase in isometric force when a high concentration (685 μm) of the D2 peptide is added to permeabilized muscles (Fig. 9). These data suggest that the linker region containing the D2 phosphorylation site mediates, to a significant extent, the tethering properties of the C-terminal portion of twitchin.

Synthetic Peptide PEVK1

This peptide is part of a short PEVK domain of twitchin (12). PEVK1 has a small inhibitory effect on force at high peptide concentrations (Fig. 9) and shows co-sedimentation with thin filaments to only a very small extent at concentrations slightly above 100 μm (Fig. 8D).

Effects of the D2 Peptide and a Combination of DX and D1 Peptides on Catch Force Maintenance

The effect of the DX and D1 peptides on catch force maintenance was determined in permeabilized muscles in which catch was induced by treatment with blebbistatin (25 μm) at pCa 6.3. Previous studies have shown that all of this force is relaxed by phosphorylation of twitchin (24). A typical trace is shown in Fig. 10A. The relative force remaining just before the addition of peptide is 0.61 ± 0.06 in the control group and 0.59 ± 0.05 in the peptide group (paired difference = 0.02 ± 0.06), showing that there is no significant difference in the decrease in force from the time the blebbistatin is added to the time of the addition of the peptides. This is consistent with there being no difference in the experimental and control groups up to that time. However, there is a significantly faster decrease in force after the addition of the DX and D1 peptides compared with the control group. This is shown by the fact that when catch force is normalized to that present just before the addition of the peptides (Fig. 10B) catch force is significantly lower (22 ± 7%, n = 7) after 30 min in the muscles treated with peptides compared with paired control muscles. These data are consistent with the idea that the peptides can compete with the thin filament binding sites on twitchin and displace it from the filament, thereby relaxing catch force. In contrast, similar experiments using the D2 peptide (Fig. 10, C and D) show no significant effect on catch force (difference = 2 ± 5%, n = 8).

FIGURE 10.
Effect of the D2 peptide and a combination of DX and D1 peptides on catch force output. Typical force traces show the design and the effect of peptides on catch force. The muscle was activated in pCa 6.3 and then put into catch by the addition of blebbistatin. ...

DISCUSSION

These results show that the N-terminal portion of twitchin containing both the DX and the D1 phosphorylation sites binds to both thin and thick filaments. The binding to thin filaments is reduced in both IGD1DXIG and DXD1 by phosphorylation (Figs. 5 and and7).7). The proteins also result in a phosphorylation-dependent increase in force in permeabilized muscles at calcium concentrations that give less than maximal force output. These data strongly suggest that the IGDXD1IG portion of twitchin participates in a linkage between thick and thin filaments that contributes to catch force maintenance and, thereby, provides a high resistance to muscle lengthening during catch. A phosphorylation-dependent binding of the C-terminal IGD2IG fragment to actin and myosin has been shown previously (26), and its possible participation as a mechanical linkage in catch has been discussed (26, 27). Overall, these results suggest that each end of the twitchin molecule acts as an independent tether between thick and thin filaments. The presence of two tethers in every twitchin molecule would provide an especially secure and redundant mechanical link between the filaments, and as such seems to be ideally suited for long term catch force maintenance and resistance to lengthening (16). Fig. 11 shows a schematic depicting the independent tethering sites between thick and thin filaments in the N- and C- terminal regions of twitchin. Of course, force maintenance by these tethers does not require any energy input. Given that there is one twitchin for every 15 myosin molecules (9), there would be a twitchin-mediated link between filaments for every 7–8 myosin cross-bridges. Importantly, phosphorylation causes a decrease in binding of both links, allowing rapid relaxation of catch force and loss of resistance to lengthening.

FIGURE 11.
Schematic showing tethers between thick and thin filaments mediated by both the N-terminal and C-terminal portions of twitchin. The top thin filament shows actin and tropomyosin (solid line). The thick filament is shown on the bottom with the myosin heads ...

Both IGDXD1IG and DXD1 show maximum binding of about one for each actin monomer in the thin filaments, but the Kd is much lower for IGDXD1IG than for DXD1 (1 versus 15 μm, respectively). This suggests that the Ig-like domains contribute to the overall binding of this portion of twitchin to the thin filament. Binding studies of GFP fusion proteins containing the three or five DFRXXL actin binding motifs of myosin light chain kinase show Kd values of about 0.25 μm (18). The higher affinity binding of these proteins compared with IGDXD1IG and DXD1 is likely, in part, to be due to the higher number of DFRXXL motifs. It must be remembered, however, that the effective concentration of IGDXD1IG in the intact muscle is higher than expected from its solution concentration because twitchin is bound to the thick filament (13, 14) and, as such, is maintained in close proximity to the thin filament.

Phosphorylation causes only a 3-fold decrease in the Kd of IGDXD1IG binding to the thin filament, whereas phosphorylation of twitchin is associated with complete relaxation of catch force. Similarly, Funabara et al. (26) showed a distinct phosphorylation-mediated decrease in binding of IGD2IG to actin, but there was still binding of the phosphorylated protein to actin. Interestingly, the unphosphorylated IGD2IG caused co-sedimentation of thin filaments with myosin thick filaments, but thiophosphorylated IGD2IG did not (26). The large effect of phosphorylation of IGD2IG on co-sedimentation of thick and thin filaments and the large effect of twitchin phosphorylation on catch force despite the persistence of some binding of phosphorylated IGD2IG and IGDXD1IG to the thin filaments is probably due to the fact that there is also a phosphorylation-dependent decrease in binding to thick filaments (Fig. 6) (26). In other words, the tethering function of twitchin is lost by phosphorylation-mediated detachment of twitchin from not only thin filaments but also thick filaments. The binding of twitchin to thick filaments is such that it remains attached during isolation of thick filaments (13, 14).3 It is not known whether sites in the D1 and D2 regions of twitchin are responsible for the tight binding of twitchin during thick filament isolation or whether the central portion of the molecule that shares domain homology with part of the A band portion of titin is responsible. In any case, the tight binding of twitchin to the thick filament is ideal for a molecule that acts as a tether between thick and thin filaments.

Both IGDXD1IG and DXD1 caused an increase in force when added to permeabilized muscles activated at pCa 6.3 where force is submaximal. Because these proteins bind both thick and thin filaments, a possible mechanism of the force increase is by tethering thick and thin filaments, just as has been proposed for the full twitchin molecule (16). The twitchin tether between filaments could form as the cycling high force myosin cross-bridge detaches from the thin filament. In such a scenario, some of the force generated by the cross-bridge could then be maintained by the tether when the cross-bridge fully detaches from the thin filament. This is equivalent to extending the duty cycle of the cross-bridge because some of the force generated by the cross-bridge is maintained by the tether when the cross-bridge detaches. The result is a higher force than expected from the number of myosin cross-bridges activated and cycling. The addition of phosphorylated IGDXD1IG does not cause an increase in force because there is a decrease in thick and thin filament binding properties that decrease the extent of tether formation. The mechanism by which unphosphorylated IGDXD1IG causes an increase in force at pCa 6.3 is likely to be similar to the mechanism by which unphosphorylated twitchin in the muscle at that calcium concentration results in a higher force output than phosphorylated twitchin (10). The similarity of this force enhancement upon the addition of IGDXD1IG to native twitchin in the muscle is striking.

The interaction of the N-terminal portion of twitchin with thin filaments was further clarified by the use of the DX and D1 peptides. Both of these peptides showed phosphorylation-dependent co-sedimentation with thin filaments (Fig. 8). The binding of the D1 peptide showed a substantially higher Kd than did DXD1 and IGDXD1IG (38, 15, and 1 μm, respectively). The actin binding site in the D1 peptide is not known, and the peptide had no effect on isometric force (Fig. 9), suggesting that it does not share a binding site with myosin. The DX peptide includes the actin binding motif DFRXXL (17, 19, 33) and causes an inhibition in force that occurs to a greater extent with the unphosphorylated than with the phosphorylated peptide (Fig. 9). This suggests that the DX peptide may bind to a region of actin that interferes with myosin cross-bridge binding and/or its transition into the high force state. It is interesting that the inhibition of force occurs with the peptide that includes the DFRXXL motif because three-dimensional image reconstruction shows that the binding of the DFRXXL motifs in smooth muscle myosin light chain kinase to actin occurs in an area of actin that should not interfere with the binding of myosin, tropomyosin, caldesmon, and calponin (34). It is possible that it is an area other than the DFRXXL motif in the DX peptide that interferes with force production.

At calcium concentrations that produce submaximal force, the mechanical link responsible for catch force maintenance can adjust during muscle shortening and subsequently maintain catch force at a shorter length (22). We have proposed that this occurs by the transition of myosin to the high force state on actin, resulting in a displacement of twitchin as a link between thick and thin filaments (24). Funabara et al. (27) have reported some experiments that support such a model for the IGD2IG region. The inhibition of force by the DX peptide suggests that this region may compete with myosin for actin binding and may play a role in mediating displacement of the IGDXD1IG region of twitchin from the thin filament resulting from high force binding of myosin to actin. A peptide derived from the myosin loop 2 region has been shown to bind to IGD2IG and to prevent the formation of a trimeric complex among thick filaments, thin filaments, and IGD2IG (27). Here, we show that this peptide also inhibits binding of thin filaments to IGDXD1IG. Because the loop 2 region of myosin is a surface loop in the actin-binding region of myosin, the loop 2 peptide inhibition of thin filament binding to IGDXD1IG suggests that the loop 2 region and IGDXD1IG compete with respect to actin binding. Because the unphosphorylated DX peptide blocks force output, it may contain a segment that binds to the region of actin to which the loop 2 region of myosin binds. These studies should be interpreted with some caution because the quantitative details of the myosin loop 2 peptide inhibition of IGDXD1IG binding to the thin filament have not been determined, and the specificity of the interaction has not been shown using a scrambled loop 2 peptide. However, the data presented here for the N-terminal portion of twitchin and in (27) for the C-terminal portion of twitchin provide support for the idea that myosin and twitchin compete for binding to actin, and it is this competition that allows activated myosin to displace twitchin from actin, detaching the twitchin tether and allowing unimpeded muscle shortening although twitchin is unphosphorylated.

Another mechanism by which activated myosin could displace twitchin is based on the work of Avrova et al. (35), showing that unphosphorylated twitchin binding to the thin filament shifts tropomyosin to a position characteristic of relaxed conditions. The authors suggest that such a mechanism might lead to unphosphorylated twitchin inhibiting the formation of high force myosin cross-bridges. On the other hand, the shifting of tropomyosin to the “on” position by the binding of myosin to the thin filament may result in the displacement of twitchin from the thin filament and loss of the tether responsible for catch.

Each actin monomer in the thin filament can bind two D2 peptides, and phosphorylation of the peptide results in the loss of one of the binding sites. It is not known if the D2 regions of two separate twitchin molecules have simultaneous access to the two different D2 peptide binding sites on the same actin monomer in the structured muscle. In any case, the persistence of binding of 1 mol of the phosphorylated D2 peptide to the actin monomer confirms that phosphorylation does not totally prevent interaction between the C-terminal portion of twitchin with actin. The portion of twitchin surrounding the D2 phosphorylation site is complicated with respect to thick and thin filament binding properties. In addition to the results presented here, Funabara et al. (27) have shown that the IGD2IG fragment of twitchin has two binding sites for a small (9-residue) actin peptide as well as two binding sites for the myosin loop 2 peptide. The interactions of all of these twitchin, actin, and myosin binding sites and their phosphorylation dependence is even more complicated, and these interactions need to be clarified with additional experiments. It is noted that the D2 peptide caused a small increase in isometric force when added to permeabilized muscles. This suggests that there is sufficient thick and thin filament binding capacity in this peptide to tether filaments and to allow some continuing force output following detachment from actin of the high force myosin cross-bridge.

The central role that the DX and D1 regions in the N-terminal portion of twitchin play in catch force maintenance is also shown by the observation that the addition of the peptides to permeabilized muscles in catch causes a significant relaxation of catch force (Fig. 10). The finding is consistent with the peptides competing with twitchin for binding to actin and in doing so loosening the tethering of twitchin to the thin filament. The D2 peptide does not cause a similar relaxation of catch force, although it does show binding to the thin filament. The lack of a loss of catch force in this case may reflect the aforementioned possibility that this peptide contains both thick and thin binding properties that would promote tethering of the thick and thin filaments.

In the peptide containing the D1 phosphorylation site, treatment with PKA results in phosphorylation of a site in addition to D1. It is likely that the phosphorylation is on serine 1067, which is the only other PKA consensus sequence in the D1 peptide. Future studies will have to determine the extent to which this site is phosphorylated in the intact muscle by activation of PKA and what the functional role might be.

Twitchin, Titin, and MyBP-C

Although the small PEVK1 peptide that is part of the PEVK region of twitchin showed very limited co-sedimentation with thin filaments, the concepts that are being developed with respect to the PEVK region of titin have analogy to the mechanism of twitchin tethering in catch muscle. The PEVK domain of titin has been shown to interact with actin (28,30, 36). There is differential binding to actin in different PEVK regions of titin, and Bianco et al. (37) suggest that the PEVK region of titin is a “promiscuous actin-binding partner,” with numerous actin binding regions, just as are present in the N- and C-terminal regions of the twitchin molecule. These actin binding sites in titin may provide a viscous load that resists the relative sliding of thick and thin filaments (36, 37). A “sticky spring” model for titin-induced force enhancement and force depression in striated muscle has been proposed in which the PEVK region interacts with the thin filament (38). Similarly, Leonard and Herzog (39) have proposed that titin is a cross-bridge force-dependent tether between thick and thin filaments. In this model, the binding of titin to the thin filament is thought to depend on active force, and the binding modifies the spring length of titin, thereby changing its contribution to total force output from the muscle.

The domain organization around the DX and D1 phosphorylation sites in twitchin shows similarity to that of cardiac MyBP-C (12). The N-terminal portion of cMyBP-C includes the domains C0-C1-m-C2 with the C0 domain and an insertion in the m-domain being specific to the cardiac isoform. The m-domain contains serine residues that can be phosphorylated by protein kinase A and also includes a DFRXXL-like actin binding motif. This region has been shown to bind to the S2 region of myosin when unphosphorylated (40) but not when phosphorylated by PKA (41). Recently, it was also shown that portions of the C0–C2 domains that include the m-domain bind thin filaments with a Kd of ~10 μm and that phosphorylation decreases the binding, probably through a change in electrostatic charge interactions (32). The striking similarities of thin and thick filament interactions in the N-terminal portion of MyBP-C and the DXD1 portion of twitchin as well as their common phosphorylation dependence suggest that MyBP-C and twitchin may share the function of a regulated tether between thick and thin filaments. Both the fact that IGDXD1IG from twitchin has a 10 fold higher affinity than does C1-m-C2 in MyBP-C and the fact that twitchin has the D2 region that can also participate as a tether suggest that the twitchin tether is optimized for the extremely prolonged force maintenance and resistance to stretch associated with the catch state. The idea of a MyBP-C-mediated tether between thick and thin filaments may seem inconsistent with the function of cardiac muscles that go through continuous rapid contraction and relaxation cycles. However, such a tether could slip with filament sliding and simply act as a regulatable viscous element during changes in muscle length. Alternatively, the interaction of MyBP-C with the thin filament may play a role in regulation by changing tropomyosin position (42) or by modifying cross-bridge kinetics by altering the interaction of myosin with actin (32). The skeletal muscle isoforms of MyBP-C show sequence homology with the cardiac isoform in the region near the DFRXXL-like motif but do not include the phosphorylation sites present in the cardiac isoform (40). The function of MyBP-C in skeletal muscle may also include being a tether between thick and thin filaments that leads to a non-regulatable viscous element that affects filament sliding.

*This work was supported, in whole or in part, by National Institutes of Health, NIAMS, Grant R01AR042758. This project was also supported in part under a grant with the Pennsylvania Department of Health.

3M. J. Siegman and T. M. Butler, unpublished observations.

2The abbreviations used are:

PKA
cyclic AMP-dependent protein kinase
ABRM
anterior byssus retractor muscle
HDTA
1,6-diaminohexane-N,N,N′,N′-tetraacetic acid.

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